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Neural-cancer interactions involve the complex interplay between the nervous system and cancer cells, influencing tumour initiation, progression, and metastasis. In gliomas, these interactions mostly entail the secretion of paracrine growth factors, and electrochemical communication mediated by synapses between neurons and glioma cells. Understanding such interactions is vital for developing new therapeutic strategies against cancer aimed at modulating neuron-to-tumour communication. For this purpose, we have used in vivo mouse models of glioblastoma (GB) and established in vitro assays to study neural-cancer interactions, including the co-culture of cancer cells with either hiPSC-derived glutamatergic neurons or GABAergic interneurons, as well as 3D cultures of tumour spheres and fetal spheroids. The co-culture of hiPSC-derived neurons and cancer cells, including GB cells, was established both as a contact and non-contact assay, allowing to study the relevance of neural-cancer interactions for cancer cell proliferation and migration. While 3D spheroids generally replicate the organization and complexity of tissues effectively, our 3D invasion assay between organ spheroids and tumour spheres enabled us to specifically examine tumour invasion. This is exemplified by GB tumour spheres that exhibit reduced invasiveness of 3D brain spheroids upon repression of EGFR regulatory sequences.

Additionally, the co-culture systems enabled us to profile the transcriptome and chromatin accessibility of GB cells upon neural activity stimulation. GB cells in contact with either glutamatergic neurons or GABAergic interneurons exhibit differential gene expression and chromatin accessibility profiles. This provides new insights into the regulatory networks mediating neuron-to-glioma communication and highlights the relevance of GABAergic signalling in GB pathogenesis. This integrated approach holds promise for furthering our understanding of neural-cancer interactions, offering potential candidates to target neural pathways involved in tumour progression. 

Background: Glioblastoma (GB) is the most aggressive of all primary brain tumours and due to its highly invasive nature, surgical resection is nearly impossible. Patients typically rely on radiotherapy with concurrent temozolomide (TMZ) treatment and face a median survival of ~ 14 months. Alterations in the Epidermal Growth Factor Receptor gene (EGFR) are common in GB tumours, but therapies targeting EGFR have not shown significant clinical efficacy.

Methods: Here, we investigated the influence of the EGFR regulatory genome on GB cells and identified novel EGFR enhancers located near the GB-associated SNP rs723527. We used CRISPR/Cas9-based approaches to target the EGFR enhancer regions, generating multiple modified GB cell lines, which enabled us to study the functional response to enhancer perturbation.

Results: Epigenomic perturbation of the EGFR regulatory region decreases EGFR expression and reduces the proliferative and invasive capacity of glioblastoma cells, which also undergo a metabolic reprogramming in favour of mitochondrial respiration and present increased apoptosis. Moreover, EGFR enhancer-perturbation increases the sensitivity of GB cells to TMZ, the first-choice chemotherapeutic agent to treat glioblastoma.

Conclusions: Our findings demonstrate how epigenomic perturbation of EGFR enhancers can ameliorate the aggressiveness of glioblastoma cells and enhance the efficacy of TMZ treatment. This study demonstrates how CRISPR/Cas9-based perturbation of enhancers can be used to modulate the expression of key cancer genes, which can help improve the effectiveness of existing cancer treatments and potentially the prognosis of difficult-to-treat cancers such as glioblastoma.

BACKGROUND: Organ culture models have been used over the past few decades to study development and disease. The in vitro three-dimensional (3D) culture system of organoids is well known, however, these 3D systems are both costly and difficult to culture and maintain. As such, less expensive, faster and less complex methods to maintain 3D cell culture models would complement the use of organoids. Chick embryos have been used as a model to study human biology for centuries, with many fundamental discoveries as a result. These include cell type induction, cell competence, plasticity and contact inhibition, which indicates the relevance of using chick embryos when studying developmental biology and disease mechanisms.

RESULTS: Here, we present an updated protocol that enables time efficient, cost effective and long-term expansion of fetal organ spheroids (FOSs) from chick embryos. Utilizing this protocol, we generated FOSs in an anchorage-independent growth pattern from seven different organs, including brain, lung, heart, liver, stomach, intestine and epidermis. These three-dimensional (3D) structures recapitulate many cellular and structural aspects of their in vivo counterpart organs and serve as a useful developmental model. In addition, we show a functional application of FOSs to analyze cell-cell interaction and cell invasion patterns as observed in cancer.

CONCLUSION: The establishment of a broad ranging and highly effective method to generate FOSs from different organs was successful in terms of the formation of healthy, proliferating 3D organ spheroids that exhibited organ-like characteristics. Potential applications of chick FOSs are their use in studies of cell-to-cell contact, cell fusion and tumor invasion under defined conditions. Future studies will reveal whether chick FOSs also can be applicable in scientific areas such as viral infections, drug screening, cancer diagnostics and/or tissue engineering.